This invention relates to a liquid chromatography device.
The field of high pressure liquid chromatography is described in M. Dong, Modern HPLC for Practising Scientists, Wiley, 2006. Briefly, chromatography is used to separate, indentify and quantify compounds from a sample consisting of a mixture of compounds. The sample is dissolved in a fluid mobile phase, which interacts with an immobile, immiscible stationary phase. In high pressure liquid chromatography (HPLC) the stationary phase is usually a column packed with particles, which maybe functionalised. The phases are chosen based on the analyte of interest's affinity towards them, relative to that of the rest of the sample. As the mobile phase moves through the stationary phase, the individual sample components will be retained by the stationary phase to varying degrees and will become separated. The retention time varies depending on the interaction strength with the stationary phase, the composition of solvent used and the flow rate of the mobile phase.
Separation power increases with smaller stationary phase particle size. However, this increases the resistance to flow making the use of high pressures desirable. High pressure liquid chromatography drives the mobile phase through columns containing particles of typical diameters 5-10 micrometers. The first HPLC pumps were capable of 500 psi, with 6000 psi typical today. Ultrahigh pressure liquid chromatography (UPLC) consists of plumbing and pumps capable of performing at 100,000 psi required to drive solvent through columns containing even smaller particles of the order of 1 micrometer diameter.
The detection of separated analytes is possible via one or more techniques, including UV-visible light absorption, fluorescence, light scattering, refractive index analysis or mass spectrometry. These techniques, particularly when used in parallel, allow the identification and absolute quantitation of a very wide range of compounds, and permit a semi-quantitative analysis of even complex, unknown analyte mixtures. Detection signals are referenced to the time of the sample's injection onto the chromatographic column: under identical conditions, a given compound will have a characteristic retention time and it is this which allows its identification. Where the detection technique is destructive the sample cannot be recovered, but in these cases it is often possible to divert a percentage of the eluent flow to a fraction collector if required.
HPLC has found wide applications in the pharmaceutical industry, environmental monitoring, medicine, academia, defence, forensic science, and elsewhere. However, use has been limited by the bulk and expense of HPLC systems. Cubic meter footprints, mains power supplies, large volumes of eluent, weight and mechanical fragility of existing HPLC systems require fixed laboratory installation. The size and the relatively low turnover of such systems have conspired to make the units extremely expensive both in terms of initial expenditure and then in terms of maintenance and servicing. Typical systems cost tens of thousands of pounds, putting them beyond the reach of all but large, well-established companies and research institutions. Furthermore, the small diameter of HPLC tubing coupled with the crude form of many of the samples analysed via this technique mean that blockages are frequent. The pressure build-up following such an event can cause significant damage to an HPLC, and even if such damage is avoided extended machine down-time is unavoidable.
The component most resistant to miniaturisation has been the pump.
For example, it is stated that a “reason for the limited interest in HPLC-like separations on chip appears to be the complexity of the plumbing since an external pump typical of standard HPLC instrumentation has to be included. The role of the chip then degrades to a capillary-like column and the advantages attributed to microfluidic devices vanish.” (Svec and Stachowiak, in Handbook of capillary and microchip electrophoresis and associated microtechniques, ed James P. Landes, CRC Press, 2008, p 1299).
The implementation of integrated microscale HPLC “has proven to be tedious, mostly for reasons related to pressure: the difficulty of generating high pressure with on-chip integrated pumps as well as of fabricating high-pressure rating microchips. Consequently, on-chip liquid chromatography is underdeveloped, not only compared to other chip-based analytical techniques but also in view of the importance of HPLC as an analytical technique.” (Khirevich et al. Anal. Chem., 2009, 81 (12), pp 4937-4945).
U.S. Pat. No. 6,572,749 notes that the problem of pumping for micro-HPLC is unsolved and teaches the use of electro-osmotic pumping. However, it only achieves 2500 psi, is dependent on lengthy columns and, in common with other electro-osmotic pumps, that there is interaction between the packing and the electro-osmotic flow.
Although the technology exists to allow the miniaturisation of stages such as detection, while complex pump systems ensure the footprint of the units remains so high there is little incentive to do so.
The invention described herein, at least in its presently preferred embodiments, addresses these and related needs.